摘要
为明确集胞藻PCC6803乙醇酸脱氢酶与水稻Rubisco小亚基叶绿体转运肽(RCTP)的融合蛋白是否能准确定位到水稻叶绿体,本研究以特异引物从集胞藻PCC6803基因组DNA中扩增获得约1.5 kb的片段,将该片段与水稻Rubisco小亚基叶绿体转运肽连接,然后将融合片段连入瞬时表达载体P322-d1-e GFP,测序,获得融合基因RCTP-gdh,进一步构建了叶绿体定位的瞬时表达载体RCTPGDH-e GFP-d1并进行了叶绿体定位研究。结果表明,集胞藻PCC6803 GDH共编码492个氨基酸,含有FAD结合域、FAD关联的氧化酶活性(222~464 aa)以及乙醇酸氧化酶亚基(51~463 aa)。激光共聚焦显微镜分析表明,融合蛋白RCTP-GDH-e GFP可以准确定位到水稻叶绿体,但其转运效率低于RCTP-e GFP的转运效率,叶绿体和细胞质中的融合蛋白RCTP-GDH-e GFP都易于聚集而呈斑点状。本研究为进一步应用集胞藻gdh基因改造C3作物光呼吸代谢途径,抑制C3作物光呼吸速率,提高作物光合速率奠定了基础。
To investigate wether the fusion protein derived from glycolate dehydrogenase in Synechocystis PCC6803 and Rubisco small subunit chloroplast transit peptide in rice could target into rice chloroplasts perfectly, a fragment about1.5Kb was amplified using specific primers from Synechocystis sp. PCC 6803 genomic DNA, the fragment was fused to transit peptide of rice Rubisco small subunit（RCTP ）, the fusion fragment RCTP-gdh was then inserted into transient expression vector P322-d1-eGFP, the sequencing results showed that we have obtained the glycolate dehydrogenase gene. Then the recombinant chloroplast-targeting transient expression vector RCTP-GDH-eGFP-d1 was constructed and itssubcellular localization was analyzed. The results showed that Synechocystis sp. PCC 6803 GDH encoded 492 amino acid residues and contained FAD binding domine, FAD-oxidase C, as well as glycolate oxidase subunit D domains.Subcellular localization analysis using confocal laser scanning microcopy （CLSM） showed that the fusion protein RCTP-GDH-eGFP could be translocated into rice chloroplasts successfully, but the targeting efficiency was lower than RCTP-eGFP and the fusion protein RCTP-GDH-eGFP tend to aggregate and form speckles in both cytoplasm and chloroplasts,which provide scientific reference to engineering the photorespiration pathway, inhibiting of photorespiration rate and increasing photosynthetic rate of C crops by further using of gdh gene.
作者
胥华伟
侯典云
XU Huawei;HOU Dianyun(College of Agriculture, Henan University of Science and Technology, Luoyang, Henan 471023)
出处
《核农学报》
CAS
CSCD
北大核心
2018年第7期1330-1337,共8页
Journal of Nuclear Agricultural Sciences
基金
国家自然科学基金(31100197)
关键词
光呼吸
集胞藻PCC6803
乙醇酸脱氢酶
叶绿体定位
瞬时表达
photorespiration
Synechocystis sp. PCC6803
glycolate dehydrogenase
chloroplast-targeting
transient
expression
